The sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol is found in the photosynthetic membranes of all plants and most photosynthetic bacteria. Progress towards the elucidation of the pathway for sulfolipid biosynthesis has been slow during the past. However, the recent isolation of sqd genes essential for sulfolipid biosynthesis in the photosynthetic bacteria R. sphaeroides, as well as in higher plants, provides promising new opportunities. Specific inactivation of the sqdD gene of R. sphaeroides resulted in the accumulation of UDP-sulfoquinovose. Inactivation of the sqdC gene led to the accumulation of a sulfur labeled compound which we tentatively identified as sulfoquinovosyl-1-O-dihydroxyacetone. This compound is most likely not a precursor of sulfolipid biosynthesis, because it is not further metabolized by wild type extracts. Based on these results we propose that the sqdC gene product interacts with the sulfoquinovosyl transferase to provide membrane association and substrate specificity for the diacylglycerol acceptor. To elucidate the role of the sqdB gene of R. sphaeroides and the corresponding gene SQD1 of A. thaliana, the respective coding sequences were cloned into expression vectors and recombinant proteins were produced in E. coli. Both enzymes were able to convert UDP-glucose at slow rates in vitro into a new compound now under investigation by MS methods.